Cardiac fibrosis is a grim consequence for almost all myocardial injuries. In myocardial infarction (MI), what starts as a protective scarring process to prevent ventricular wall rupture becomes a pathological remodeling of the tissue with the accumulation of excess extracellular matrix (ECM) proteins. Eventually, this adaptation impedes the mechanical and electrical properties of the myocardium resulting in heart failure. Recently, we showed that periostin (Postn) expressing cells that arise from resident cardiac fibroblasts (CFs) are a potential therapeutic target since they differentiate into the scar associated, matrix-producing cell-type after MI injury. In fact, deletion of these cells after an acute MI injury eliminates interstitial fibrosis but results in ventricular rupture which is a hallmark outcome of impaired ECM deposition during the acute phase of MI. However, if we delete these cells during a chronic injury such as pressure overload-induced cardiac fibrosis model, we detect sustained perivascular fibrosis. Previous studies also report heterogeneity of origin and function for ECM-producing cells associated with different cardiac diseases. Consequently, our inability to identify cell- and state-specific therapeutic targets render cardiac fibrosis yet an incurable disease. Therefore, there is a critical need to determine the cellular composition and functional heterogeneity within ECM-producing fibroblasts. Until very recently, the main limitation has been the inability to accurately interrogate and manipulate the activities of different CF sub-populations differentiated from cells, including pericytes, endothelial cells, resident inflammatory cells in vivo given a lack of cell type-specific genetic tools. Recently, we and others have generated several novel genetic tools that now allow us to investigate all of the matrix-producing cells and their activated forms. Utilizing these new genetic tools in lineage tracing, gain-of-function, and loss-of-function studies, we will interrogate and determine the origin and function of all ECM-producing cell types as well as the molecular mechanisms that regulate CF sustained pathological activation and differentiation after acute or chronic disease models in mice. Our recent work where we effectively interrogated Postn expressing CF lineage in comparison to Postn negative CFs in a single-cell RNA sequencing analysis revealed distinct gene expression profiles between these two populations. Depending on the injury type, such as hearts subjected to MI, TAC, or Angiotensin induced fibrosis, we observed differences in ECM components as well as cellular composition. Finally, our preliminary data showed here identify another cell lineage that involves perivascular fibrosis. Therefore, we hypothesize that pathological ECM deposition resulting in fibrosis comes from disease-specific specialized sub-populations of CFs with distinct gene expressions. The following aims will rigorously interrogate CF subpopulations and the molecular mechanisms that regulate CF activation and ECM composition.